Methods for preventing or treating mitochondrial permeability transition

ABSTRACT

The invention provides a method of reducing or preventing mitochondrial permeability transitioning. The method comprises administering an effective amount of an aromatic-cationic peptide having at least one net positive charge; a minimum of four amino acids; a maximum of about twenty amino acids; a relationship between the minimum number of net positive charges (p m ) and the total number of amino acid residues (r) wherein 3p m  is the largest number that is less than or equal to r+1; and a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (p t ) wherein 2 a is the largest number that is less than or equal to p t +1, except that when a is 1, p t  may also be 1.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/427,804, filed on Jun. 30, 2006, which is a continuation-in-part ofU.S. application Ser. No. 10/771,232, filed on Feb. 3, 2004, whichclaims priority to U.S. Provisional Application Ser. No. 60/444,777,filed on Feb. 4, 2003, and U.S. Provisional Application No. 60/535,690,filed on Jan. 8, 2004, the contents of which are hereby incorporated byreference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support from the NationalInstitute on Drug Abuse under Grant No. POI DA08924-08. The U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Mitochondria exist in virtually all eukaryotic cells, and are essentialto cell survival by producing adenosine triphosphate (ATP) via oxidativephosphorylation. Interruption of this vital function can lead to celldeath.

Mitochondria also play a major role in intracellular calcium regulationby accumulating calcium (Ca²⁺). Accumulation of calcium occurs in themitochondria) matrix through a membrane potential-driven uniporter.

The uptake of calcium activates mitochondrial dehydrogenases, and may beimportant in sustaining energy production and oxidative phosphorylation.In addition, the mitochondria serve as a sink for excessive cytosolicCa²⁺, thus protecting the cell from Ca²⁺ overload and necrotic death.

Ischemia or hypoglycemia can lead to mitochondrial dysfunction,including ATP hydrolysis and Ca²⁺ overload. The dysfunction causesmitochondrial permeability transition (MPT). MPT is characterized byuncoupling of oxidative phosphorylation, loss of mitochondrial membranepotential, increased permeability of the inner membrane and swelling.

In addition, the mitochondria intermembrane space is a reservoir ofapoptogenic proteins. Therefore, the loss of mitochondrial potential andMPT can lead to release of apoptogenic proteins into the cytoplasm. Notsurprisingly, there is accumulating evidence that MPT is involved innecrotic and apoptotic cell death (Crompton, Biochem J., 341:233-249(1999)). Milder forms of cellular insult may lead to apoptosis ratherthan necrosis.

Cyclosporin can inhibit MPT. Blockade of MPT by cyclosporin A caninhibit apoptosis in several cell types, including cells undergoingischemia, hypoxia, Ca²⁺ overload and oxidative stress (Kroemer et al.,Annu Rev Physiol., 60:619-642 (1998)).

Cyclosporin A, however, is less than optimal as a treatment drug againstnecrotic and apoptotic cell death. For example, cyclosporin A does notspecifically target the mitochondria. In addition, it is poorlydelivered to the brain. Furthermore, the utility of cyclosporin A isreduced by its immunosuppressant activity.

The tetrapeptide [Dmt¹]DALDA (2′,6′-dimethyltyrosine-D-Arg-Phe-Lys-NH₂,SS-02) has a molecular weight of 640 and carries a net three positivecharge at physiological pH. [Dmt¹]DALDA readily penetrates the plasmamembrane of several mammalian cell types in an energy-independent manner(Zhao et al., J Pharmacol Exp Ther., 304:425-432, 2003) and penetratesthe blood-brain barrier (Zhao et al., J Pharmacol Exp Ther.,302:188-196, 2002). Although [Dmt¹]DALDA has been shown to be a potentmu-opioid receptor agonist, its utility has not been expanded to includethe inhibition of MPT.

Thus, there is a need to inhibit MPT in conditions such asischemia-reperfusion, hypoxia, hypoglycemia, and other diseases andconditions which result in pathological changes as a result of thepermeability transitioning of the mitochondrial membranes. Such diseasesand conditions include many of the common neurodegenerative diseases.

SUMMARY OF THE INVENTION

These and other objectives have been met by the present invention whichprovides a method for reducing the number of mitochondria undergoing amitochondria permeability transition (MPT), or preventing mitochondrialpermeability transitioning in any mammal that has need thereof. Themethod comprises administering to the mammal an effective amount of anaromatic-cationic peptide having:

a. at least one net positive charge;

b. a minimum of three amino acids;

c. a maximum of about twenty amino acids;

d. a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and

e. a relationship between the minimum number of aromatic groups (a) andthe total number of net positive charges (p_(t)) wherein 2a is thelargest number that is less than or equal to p_(t)+1, except that when ais 1, p_(t) may also be 1.

In another embodiment, the invention provides a method for reducing thenumber of mitochondria undergoing a mitochondrial permeabilitytransition (MPT), or preventing mitochondrial permeability transitioningin a removed organ of a mammal. The method comprises administering tothe removed organ an effective amount of an aromatic-cationic peptidehaving:

a. at least one net positive charge;

b. a minimum of three amino acids;

c. a maximum of about twenty amino acids;

d. a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and

e. a relationship between the minimum number of aromatic groups (a) andthe total number of net positive charges (p_(t)) wherein 2a is thelargest number that is less than or equal to p_(t)+1, except that when ais 1, p_(t) may also be 1.

In yet another embodiment, the invention provides a method of reducingthe number of mitochondria undergoing mitochondrial permeabilitytransition (MPT), or preventing mitochondria) permeability transitioningin a mammal in need thereof. The method comprises administering to themammal an effective amount of an aromatic-cationic peptide having:

a. at least one net positive charge;

b. a minimum of three amino acids;

c. a maximum of about twenty amino acids;

d. a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3 p_(m)is the largest number that is less than or equal to r+1; and

e. a relationship between the minimum number of aromatic groups (a) andthe total number of net positive charges (p_(t)) wherein 3a is thelargest number that is less than or equal to p_(t)+1, except that when ais 1, p_(t) may also be 1.

In a further embodiment, the invention provides a method of reducing thenumber of mitochondria undergoing mitochondrial permeability transition(MPT), or preventing mitochondrial permeability transitioning in aremoved organ of a mammal. The method comprises administering to theremoved organ an effective amount of an aromatic-cationic peptidehaving:

a. at least one net positive charge;

b. a minimum of three amino acids;

c. a maximum of about twenty amino acids;

d. a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and

e. a relationship between the minimum number of aromatic groups (a) andthe total number of net positive charges (p_(t)) wherein 3a is thelargest number that is less than or equal to p_(t)+1, except that when ais 1, p_(t) may also be 1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Cellular internalization and accumulation of [Dmt¹] DALDA(SS-02) in mitochondria. (FIG. 1A) Mitochondrial uptake of SS-19 wasdetermined using fluorescence spectrophotometry (ex/em=320/420 nm).Addition of isolated mouse liver mitochondria (0.35 mg/ml) resulted inimmediate quenching of SS-19 fluorescence intensity (gray line).Pretreatment of mitochondria with FCCP (1.5 μM) reduced quenching by<20% (black line). (FIG. 1B) Isolated mitochondria were incubated with[³H]SS-02 at 37° C. for 2 min. Uptake was stopped by centrifugation(16000×g) for 5 min at 4° C., and radioactivity determined in thepellet. Pretreatment of mitochondria with FCCP inhibited [³H]SS-02uptake by ˜20%. Data are shown as mean±s.e.; n=3. *, P<0.05 by Student'st-test. (FIG. 1C) Uptake of TMRM by isolated mitochondria is lost uponmitochondrial swelling induced by alamethicin, while uptake of SS-19 isretained to a large extent. Black line, TMRM; red line, SS-19. (FIG. 1D)Addition of SS-02 (200 μM) to isolated mitochondria did not altermitochondrial potential, as measured by TMRM fluorescence. Addition ofFCCP (1.5 μM) caused immediate depolarization while Ca²⁺ (150 μM)resulted in depolarization and progressive onset of MPT.

FIG. 2. [Dmt¹]DALDA (SS-02) protects against mitochondrial permeabilitytransition (MPT) induced by Ca²⁺ overload and 3-nitroproprionic acid(3NP). (FIG. 2A) Pretreatment of isolated mitochondria with 10 μM SS-02(addition indicated by down arrow) prevented onset of MPT caused by Ca²⁺overload (up arrow). Black line, buffer; red line, SS-02 (FIG. 2B)Pretreatment of isolated mitochondria with SS-02 increased mitochondrialtolerance of multiple Ca²⁺ additions prior to onset of MPT. Arrowindicates addition of buffer or SS-02. Line 1, buffer; line 2, 50 μMSS-02; line 3, 100 μM SS-02. (FIG. 2C) SS-02 dose-dependently delayedthe onset of MPT caused by 1 mM 3NP. Arrow indicates addition of bufferor SS-02. Line 1, buffer; line 2, 0.5 μM SS-02; line 3, 5 μM SS-02; line4, 50 μM SS-02.

FIG. 3. [Dmt¹]DALDA (SS-02) inhibits mitochondrial swelling andcytochrome c release. (FIG. 3A) Pretreatment of isolated mitochondriawith SS-02 dose dependently inhibited mitochondrial swelling induced by200 μM Ca²⁺ in a dose-dependent manner. Swelling was measured byabsorbance at 540 nm. (FIG. 3B) SS-02 inhibited Ca²⁺-induced release ofcytochrome c from isolated mitochondria. The amount of cytochrome creleased was expressed as percent of total cytochrome c in mitochondria.Data are presented as mean±s.e., n=3. (FIG. 3C) SS-02 also inhibitedmitochondrial swelling induced by MPP⁺ (300 μM).

FIG. 4. D-Arg-Dmt-Lys-Phe-NH₂ (SS-31) inhibits mitochondrial swellingand cytochrome c release. (FIG. 4A) Pretreatment of isolatedmitochondria with SS-31 (10 μM) prevents onset of MPT induced by Ca²⁺.Gray line, buffer; red line, SS-31. (FIG. 4B) Pretreatment ofmitochondria with SS-31 (50 μM) inhibited mitochondrial swelling inducedby 200 mM Ca²⁺. Swelling was measured by light scattering measured at570 nm. (FIG. 4C). Comparison of SS-02 and SS-31 with cyclosporine (CsA)in inhibiting mitochondrial swelling and cytochrome c release induced byCa²⁺, The amount of cytochrome c released was expressed as percent oftotal cytochrome c in mitochondria. Data are presented as mean±s.e.,n=3.

FIG. 5. [Dmt¹]DALDA (SS-02) and D-Arg-Dmt-Lys-Phe-NH₂ (SS-31) protectsmyocardial contractile force during ischemia-reperfusion in the isolatedperfused guinea pig heart. Hearts were perfused with buffer or buffercontaining SS-02 (100 nM) or SS-31 (1 nM) for 30 min and then subjectedto 30-min global ischemia. Reperfusion was carried out using the sameperfusion solution. Significant differences were found among the threetreatment groups (2-way ANOVA, P<0.001).

FIG. 6. Addition of [Dmt¹]DALDA to cardioplegic solution significantlyenhanced contractile function after prolonged ischemia in the isolatedperfused guinea pig heart. After 30 min stabilization, hearts wereperfused with St. Thomas cardioplegic solution (CPS) or CPS containing[Dmt¹]DALDA at 100 μm for 3 min. Global ischemia was then induced bycomplete interruption of coronary perfusion for 90 min. Reperfusion wassubsequently carried out for 60 min with oxygenated Krebs-Henseleitsolution. Post-ischemic contractile force was significantly improved inthe group receiving [Dmt¹]DALDA (P<0.001).

FIG. 7. SS-31 protected against tBHP-induced mitochondrialdepolarization and viability. N₂A cells were plated in glass bottomdishes and treated with (A) control, or with (B) 50 μM tBHP, alone orwith (e) 1 nM SS-31, for 6 hours. Cells were loaded with TMRM (20 nM)and imaged by confocal laser scanning microscopy using ex/em of 552/570nm.

FIG. 8. Representative rat heart slices stained; (a) is myocardialischemia area at risk, as determined by Evans blue dye (unstained byblue dye), and (b) is infarct myocardium as determined by TTC stainingand formalin fixation (the pink and white areas unstained by TTC).

FIG. 9. The area at risk relative to the left ventricle after 1 hourischemia followed by 1 hour of reperfusion in rats treated with control,SS-31 or SS-20 administered 30 min before ligation and 5 min beforereperfusion. Bars represent group mean, brackets indicated S.E.M.

FIG. 10. Infarct size relative to the left ventricle after 1 hourischemia followed by 1 hour of reperfusion in rats treated with control,SS-31 or SS-20 administered 30 min before ligation and 5 min beforereperfusion. Bars represent group mean, brackets indicate S.E.M.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the surprising discovery by the inventors thatcertain aromatic-cationic peptides significantly reduce the number ofmitochondria undergoing, or even completely preventing, mitochondrialpermeability transition (MPT). Reducing the number of mitochondriaundergoing, and preventing, MPT is important, since MPT is associatedwith several common diseases and conditions in mammals. In addition, aremoved organ of a mammal is susceptible to MPT. These diseases andconditions are of particular clinical importance as they afflict a largeproportion of the human population at some stage during their lifetime.

Peptides

The aromatic-cationic peptides useful in the present invention arewater-soluble and highly polar. Despite these properties, the peptidescan readily penetrate cell membranes.

The aromatic-cationic peptides useful in the present invention include aminimum of three amino acids, and preferably include a minimum of fouramino acids, covalently joined by peptide bonds.

The maximum number of amino acids present in the aromatic-cationicpeptides of the present invention is about twenty amino acids covalentlyjoined by peptide bonds. Preferably, the maximum number of amino acidsis about twelve, more preferably about nine, and most preferably aboutsix. Optimally, the number of amino acids present in the peptides isfour.

The amino acids of the aromatic-cationic peptides useful in the presentinvention can be any amino acid. As used herein, the term “amino acid”is used to refer to any organic molecule that contains at least oneamino group and at least one carboxyl group. Preferably, at least oneamino group is at the a position relative to the carboxyl group.

The amino acids may be naturally occurring. Naturally occurring aminoacids include, for example, the twenty most common levorotatory (L,)amino acids normally found in mammalian proteins, i.e., alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Glu), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan, (Trp), tyrosine (Tyr), and valine (Val).

Other naturally occurring amino acids include, for example, amino acidsthat are synthesized in metabolic processes not associated with proteinsynthesis. For example, the amino acids ornithine and citrulline aresynthesized in mammalian metabolism during the production of urea.

The peptides useful in the present invention can contain one or morenonnaturally occurring amino acids. The non-naturally occurring aminoacids may be L-, dextrorotatory (D), or mixtures thereof. Optimally, thepeptide has no amino acids that are naturally occurring.

Non-naturally occurring amino acids are those amino acids that typicallyare not synthesized in normal metabolic processes in living organisms,and do not naturally occur in proteins. In addition, the non-naturallyoccurring amino acids useful in the present invention preferably arealso not recognized by common proteases.

The non-naturally occurring amino acid can be present at any position inthe peptide. For example, the non-naturally occurring amino acid can beat the N terminus, the C-terminus, or at any position between theN-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups. Some examples of alkyl amino acids includea-aminobutyric acid, (aminobutyric acid, y-aminobutyric acid,6-aminovaleric acid, and E-aminocaproic acid. Some examples of arylamino acids include ortho-, meta, and para-aminobenzoic acid. Someexamples of alkylaryl amino acids include ortho-, meta-, and paraaminophenyl acetic acid, and y-phenyl-R-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives ofnaturally occurring amino acids. The derivatives of naturally occurringamino acids may, for example, include the addition of one or morechemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva), norleucine (Nle), and hydroxyproline (Hyp).

Another example of a modification of an amino acid in a peptide usefulin the methods of the present invention is the derivatization of acarboxyl group of an aspartic acid or a glutamic acid residue of thepeptide. One example of derivatization is amidation with ammonia or witha primary or secondary amine, e.g. methylamine, ethylamine,dimethylamine or dethylamine. Another example of derivatization includesesterification with, for example, methyl or ethyl alcohol.

Another such modification includes derivatization of an amino group of alysine, arginine, or histidine residue. For example, such amino groupscan be acylated. Some suitable acyl groups include, for example, abenzoyl group or an alkanoyl group comprising any of the C₁-C₄ alkylgroups mentioned above, such as an acetyl or propionyl group.

The non-naturally occurring amino acids are preferably resistant, andmore preferably insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD non-naturally occurring amino acids. The D-amino acids do not normallyoccur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell, as used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides useful in themethods of the invention should have less than five, preferably lessthan four, more preferably less than three, and most preferably, lessthan two contiguous L-amino acids recognized by common proteases,irrespective of whether the amino acids are naturally or non-naturallyoccurring. Optimally, the peptide has only D-amino acids, and no L-aminoacids.

If the peptide contains protease sensitive sequences of amino acids, atleast one of the amino acids is preferably a non-naturally-occurringv-amino acid, thereby conferring protease resistance. An example of aprotease sensitive sequence includes two or more contiguous basic aminoacids that are readily cleaved by common proteases, such asendopeptidases and trypsin. Examples of basic amino acids includearginine, lysine and histidine.

It is important that the aromatic-cationic peptides have a minimumnumber of net positive charges at physiological pH in comparison to thetotal number of amino acid residues in the peptide. The minimum numberof net positive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r).

The minimum number of net positive charges discussed below are all atphysiological pH. The term “physiological pH” as used herein refers tothe normal pH in the cells of the tissues and organs of the mammalianbody. For instance, the physiological pH of a human is normallyapproximately 7.4, but normal physiological pH in mammals may be any pHfrom about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-Arg has one negatively chargedamino acid (i.e., Glu) and four positively charged amino acids (i.e.,two Arg residues, one Lys, and one His). Therefore, the above peptidehas a net positive charge of three.

In one embodiment of the present invention, the aromatic-cationicpeptides have a relationship between the minimum number of net positivecharges at physiological pH (p_(m)) and the total number of amino acidresidues (r) wherein 3 p_(m) is the largest number that is less than orequal to r+1. In this embodiment, the relationship between the minimumnumber of net positive charges (p_(m)) and the total number of aminoacid residues (r) is as follows:

(r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 33 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2 p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

(r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 55 6 6 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, preferably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a).

Naturally occurring amino acids that have an aromatic group include theamino acids histidine, tryptophan, tyrosine, and phenylalanine. Forexample, the hexapeptide Lys-Gln-Tyr-Arg-Phe-Trp has a net positivecharge of two (contributed by the lysine and arginine residues) andthree aromatic groups (contributed by tyrosine, phenylalanine andtryptophan residues).

In one embodiment of the present invention, the aromatic-cationicpeptides useful in the methods of the present invention have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges at physiological pH (p_(t)) wherein3a is the largest number that is less than or equal to p_(t)+1, exceptthat when p_(t) is 1, a may also be 1. In this embodiment, therelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) is as follows:

(p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (a) 1 1 1 1 22 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment the aromatic-cationic peptides have a relationshipbetween the minimum number of aromatic groups (a) and the total numberof net positive charges (p_(t)) wherein 2a is the largest number that isless than or equal to p_(t)+1. In this embodiment, the relationshipbetween the minimum number of aromatic amino acid residues (a) and thetotal number of net positive charges (p_(t)) is as follows:

(p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (a) 1 1 2 2 33 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are preferably amidated with, for example, ammonia to formthe C-terminalamide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-dethyl amido, Nmethyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group.

The free carboxylate groups of the asparagine, glutamine, aspartic acid,and glutamic acid residues not occurring at the C-terminus of thearomatic-cationic peptides of the present invention may also be amidatedwherever they occur within the peptide. The amidation at these internalpositions may be with ammonia or any of the primary or secondary aminesdescribed above.

In one embodiment, the aromatic-cationic peptide useful in the methodsof the present invention is a tripeptide having two net positive chargesand at least one aromatic amino acid. In a particular embodiment, thearomatic-cationic peptide useful in the methods of the present inventionis a tripeptide having two net positive charges and two aromatic aminoacids.

Aromatic-cationic peptides useful in the methods of the presentinvention include, but are not limited to, the following peptideexamples:

-   Lys-D-Arg-Tyr-NH₂,-   Phe-D-Arg-His,-   D-Tyr-Trp-Lys-NH₂,-   Trp-D-Lys-Tyr-Arg-NH₂,-   Tyr-His-D-Gly-Met,-   Phe-Arg-D-His-Asp,-   Tyr-D-Arg-Phe-Lys-Glu-NH₂,-   Met-Tyr-D-Lys-Phe-Arg,-   D-His-Glu-Lys-Tyr-D-Phe-Arg,-   Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂,-   Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His,-   Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂,-   Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂,-   Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys,-   Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂,-   Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys,-   Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂,-   D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH₂,-   Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe,-   Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe,-   Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂,-   Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr,-   Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys,    Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH₂,-   Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-Gly,-   D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂,-   Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe,-   His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂,-   Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp,    and-   Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂.

In one embodiment, the peptides useful in the methods of the presentinvention have mu-opioid receptor agonist activity (i.e., activate themu-opioid receptor). Activation of the mu-opioid receptor typicallyelicits an analgesic effect.

In certain instances, an aromatic-cationic peptide having mu-opioidreceptor activity is preferred. For example, during short-termtreatment, such as in an acute disease or condition, it may bebeneficial to use an aromatic-cationic peptide that activates themu-opioid receptor. Such acute diseases and conditions are oftenassociated with moderate or severe pain. In these instances, theanalgesic effect of the aromatic cationic peptide may be beneficial inthe treatment regimen of the patient or other mammal, although anaromatic-cationic peptide which does not activate the mu-opioid receptormay also be used with or without an analgesic according to clinicalrequirements.

Alternatively, in other instances, an aromatic-cationic peptide thatdoes not have mu-opioid receptor activity is preferred. For example,during long-term treatment, such as in a chronic disease state orcondition, the use of an aromatic-cationic peptide that activates themu-opioid receptor may be contraindicated. In these instances thepotentially adverse or addictive effects of the aromatic-cationicpeptide may preclude the use of an aromatic-cationic peptide thatactivates the mu-opioid receptor in the treatment regimen of a humanpatient or other mammal.

Potential adverse effects may include sedation, constipation andrespiratory depression. In such instances an aromatic-cationic peptidethat does not activate the mu-opioid receptor may be an appropriatetreatment.

Examples of acute conditions include heart attack, stroke and traumaticinjury. Traumatic injury may include traumatic brain and spinal cordinjury.

Examples of chronic diseases or conditions include coronary arterydisease and any neurodegenerative disorders, such as those describedbelow.

Peptides useful in the methods of the present invention which have muopioid receptor activity are typically those peptides which have atyrosine residue or a tyrosine derivative at the N-terminus (i.e., thefirst amino acid position). Preferred derivatives of tyrosine include2′-methyltyrosine (Mmt); 2′,6′-dimethyltyrosine (2′6′Dmt);3′,5′-dimethyltyrosine (3′5′Dmt); N,2′,6′-trimethyl tyrosine (Tmt); and2′hydroxy-6′-methyltryosine (Hmt).

In a particular preferred embodiment, a peptide that has mu-opioidreceptor activity has the formula Tyr-D-Arg-Phe-Lys-NH₂ (for conveniencerepresented by the acronym: DALDA, which is referred to herein asSS-01). DALDA has a net positive charge of three, contributed by theamino acids tyrosine, arginine, and lysine and has two aromatic groupscontributed by the amino acids phenylalanine and tyrosine. The tyrosineof DALDA can be a modified derivative of tyrosine such as in 2′,6′dimethyltyrosine to produce the compound having the formula2′,6′-Dmt-D-Arg-PheLys-NH₂ (i.e., Dmt¹-DALDA, which is referred toherein as SS-02).

Peptides that do not have mu-opioid receptor activity generally do nothave a tyrosine residue or a derivative of tyrosine at the N-terminus(i.e., amino acid position one). The amino acid at the N-terminus can beany naturally occurring or nonnaturally occurring amino acids other thantyrosine.

In one embodiment, the amino acid at the N-terminus is phenylalanine orits derivative. Preferred derivatives of phenylalanine include 2′-methylphenylalanine (Mmp), 2′,6′-dimethylphenylalanine (Dmp),N,2′,6′-trimethylphenylalanine (Tmp), and2′-hydroxy-6′-methylphenylalanine (Hmp).

Other aromatic-cationic peptide that does not have mu-opioid receptoractivity has the formula Phe-D-Arg-Phe-Lys-NH₂ (i.e., Phe¹-DALDA, whichis referred to herein as SS-20). Alternatively, the N-terminalphenylalanine can be a derivative of phenylalanine such as2′,6′-dimethylphenylalanine (2′6′Dmp). DALDA containing2′,6′-dimethylphenylalanine at amino acid position one has the formula2′,6′-Dmp-D-Arg-Phe-Lys-NH₂ (i.e., 2′6′Dmp¹-DALDA).

In a preferred embodiment, the amino acid sequence of Dmt¹-DALDA (SS-02)is rearranged such that Dint is not at the N-terminus. An example ofsuch an aromatic-cationic peptide that does not have mu-opioid receptoractivity has the formula D-Arg-2′6′Dmt-Lys-Phe-NH₂ (referred to in thisspecification as SS-31).

DALDA, Phe¹-DALDA, SS-31, and their derivatives can further includefunctional analogs. A peptide is considered a functional analog ofDALDA, Phe¹-DALDA, or SS-31 if the analog has the same function asDALDA, Phe¹-DALDA, or SS-31. The analog may, for example, be asubstitution variant of DALDA, Phe¹-DALDA, or SS-31, wherein one or moreamino acid is substituted by another amino acid.

Suitable substitution variants of DALDA, Phe¹-DALDA, or SS-31 includeconservative amino acid substitutions. Amino acids may be groupedaccording to their physicochemical characteristics as follows:

a. Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);

b. Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

c. Basic amino acids: His(H) Arg(R) Lys(K);

d. Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

e. Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His(H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

Examples of analogs useful in the practice of the present invention thatactivate mu-opioid receptors include, but are not limited, to thearomatic-cationic peptides shown in Table 1.

TABLE 1 Amino Acid Amino Acid Amino Acid Amino Acid Amino Acid Position5 C-Terminal Position 1 Position 2 Position 3 Position 4 (if present)Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ Tyr D-Arg PheDab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂ 2′6′Dmt D-ArgPhe Lys Cys NH₂ 2′6′Dmt D-Arg Phe Lys-NH(CH₂)_(2—)NH- NH₂ dns 2′6′DmtD-Arg Phe Lys-NH(CH₂)_(2—)NH- NH₂ atn 2′6′Dmt D-Arg Phe dnsLys NH₂2′6′Dmt D-Cit Phe Lys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg PheOrn NH₂ 2′6′Dmt D-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′DmtD-Arg Phe Ahp (2- NH₂ aminoheptanoic acid) Bio-2′6′Dmt D-Arg Phe Lys NH₂3′5′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg PheDab NH₂ 3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg TyrOrn NH₂ Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg TyrLys NH₂ 2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′DmtD-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′DmtOrn NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂3′5Dmt D-Arg 3′5Dmt Arg NH₂ 3′5Dmt D-Arg 3′5Dmt Lys NH₂ 3′5Dmt D-Arg3′5Dmt Orn NH₂ 3′5Dmt D-Arg 3′5Dmt Dab NH₂ Tyr D-Lys Phe Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂ 2′6′DmtD-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′Dmt D-Lys PheDab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ Tyr D-LysTyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ Tyr D-Lys TyrDap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂ 2′6′DmtD-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys 2′6′Dmt LysNH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dab NH₂ 2′6′DmtD-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′Dmt D-Arg PheatnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′Dmt Orn NH₂3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NH₂ Tyr D-LysPhe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dab Phe Arg NH₂ Tyr D-Dap PheArg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Orn Phe Arg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂3′5′Dmt D-Arg Phe Arg NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ 3′5′Dmt D-Orn PheArg NH₂ Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn Tyr Arg NH₂ Tyr D-Dab Tyr ArgNH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt ArgNH₂ 3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′DmtD-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe LysNH₂ Mmt D-Arg Phe Orn NH₂ Mmt D-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂Tmt D-Arg Phe Lys NH₂ Tmt D-Arg Phe Orn NH₂ Tmt D-Arg Phe Dab NH₂ TmtD-Arg Phe Dap NH₂ Hmt D-Arg Phe Lys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-ArgPhe Dab NH₂ Hmt D-Arg Phe Dap NH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys PheOrn NH₂ Mmt D-Lys Phe Dab NH₂ Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe ArgNH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂Tmt D-Lys Phe Dap NH₂ Tmt D-Lys Phe Arg NH₂ Hmt D-Lys Phe Lys NH₂ HmtD-Lys Phe Orn NH₂ Hmt D-Lys Phe Dab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-LysPhe Arg NH₂ Mmt D-Lys Phe Arg NH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab PheArg NH₂ Mmt D-Dap Phe Arg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe ArgNH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂Tmt D-Arg Phe Arg NH₂ Hmt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ HmtD-Dab Phe Arg NH₂ Hmt D-Dap Phe Arg NH₂ Hmt D-Arg Phe Arg NH₂ Dab =diaminobutyric Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt =2′-methyltyrosine Tmt = N,2′6′-trimethyltyrosine Hmt =2′hydroxy,6′-methyltyrosine dnsDap = β-dansyl-L-α,β-diaminopropionicacid antDap = β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of analogs useful in the practice of the present invention thatdo not activate mu-opioid receptors include, but are not limited to, thearomatic-cationic peptides shown in Table 2.

TABLE 2 Amino Acid Amino Acid Amino Acid Amino Acid C-Terminal Position1 Position 2 Position 3 Position 5 Modification D-Arg Dmt Lys Phe NH₂D-Arg Dmt Phe Lys NH₂ D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-ArgLys Dmt Phe NH₂ D-Arg Lys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe LysD-Arg Dmt NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl

The amino acids of the peptides shown in table 1 and 2 may be in eitherthe L- or the D-configuration.

Methods of Treating

The peptides described above are useful in treating any disease orcondition that is associated with MPT. Such diseases and conditionsinclude, but are not limited to, ischemia and/or reperfusion of a tissueor organ, hypoxia and any of a number of neurodegenerative diseases.Mammals in need of treatment or prevention of MPT are those mammalssuffering from these diseases or conditions.

Ischemia in a tissue or organ of a mammal is a multifaceted pathologicalcondition which is caused by oxygen deprivation (hypoxia) and/or glucose(e.g., substrate) deprivation. Oxygen and/or glucose deprivation incells of a tissue or organ leads to a reduction or total loss of energygenerating capacity and consequent loss of function of active iontransport across the cell membranes. Oxygen and/or glucose deprivationalso leads to pathological changes in other cell membranes, includingpermeability transition in the mitochondrial membranes. In additionother molecules, such as apoptotic proteins normally compartmentalizedwithin the mitochondria, may leak out into the cytoplasm and causeapoptotic cell death. Profound ischemia can lead to necrotic cell death.

Ischemia or hypoxia in a particular tissue or organ may be caused by aloss or severe reduction in blood supply to the tissue or organ. Theloss or severe reduction in blood supply may, for example, be due tothromboembolic stroke, coronary atherosclerosis, or peripheral vasculardisease. The tissue affected by ischemia or hypoxia is typically muscle,such as cardiac, skeletal, or smooth muscle.

The organ affected by ischemia or hypoxia may be any organ that issubject to ischemia or hypoxia. Examples of organs affected by ischemiaor hypoxia include brain, heart, kidney, and prostate. For instance,cardiac muscle ischemia or hypoxia is commonly caused by atheroscleroticor thrombotic blockages which lead to the reduction or loss of oxygendelivery to the cardiac tissues by the cardiac arterial and capillaryblood supply. Such cardiac ischemia or hypoxia may cause pain andnecrosis of the affected cardiac muscle, and ultimately may lead tocardiac failure.

Ischemia or hypoxia in skeletal muscle or smooth muscle may arise fromsimilar causes. For example, ischemia or hypoxia in intestinal smoothmuscle or skeletal muscle of the limbs may also be caused byatherosclerotic or thrombotic blockages.

Reperfusion is the restoration of blood flow to any organ or tissue inwhich the flow of blood is decreased or blocked. For example, blood flowcan be restored to any organ or tissue affected by ischemia or hypoxia.The restoration of blood flow (reperfusion) can occur by any methodknown to those in the art. For instance, reperfusion of ischemic cardiactissues may arise from angioplasty, coronary artery bypass graft, or theuse of thrombolytic drugs.

The methods of the present invention can also be used in the treatmentor prophylaxis of neurodegenerative diseases associated with MPT.Neurodegenerative diseases associated with MPT include, for instance,Parkinson's disease, Alzheimer's disease, Huntington's disease andAmyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig's disease).The methods of the present invention can be used to delay the onset orslow the progression of these and other neurodegenerative diseasesassociated with MPT. The methods of the present invention areparticularly useful in the treatment of humans suffering from the earlystages of neurodegenerative diseases associated with MPT and in humanspredisposed to these diseases.

The peptides useful in the present invention may also be used inpreserving an organ of a mammal prior to transplantation. For example, aremoved organ can be susceptible to MPT due to lack of blood flow.Therefore, the peptides can be used to prevent MPT in the removed organ.

The removed organ can be placed in a standard buffered solution, such asthose commonly used in the art. For example, a removed heart can beplaced in a cardioplegic solution containing the peptides describedabove. The concentration of peptides in the standard buffered solutioncan be easily determined by those skilled in the art. Suchconcentrations may be, for example, between about 0.1 nM to about 10 μM,preferably about 1 μM to about 10 μM.

The peptides may also be administered to a mammal taking a drug to treata condition or disease. If a side effect of the drug includes MPT,mammals taking such drugs would greatly benefit from the peptides of theinvention.

An example of a drug which induces cell toxicity by effecting MPT is thechemotherapy drug Adriamycin.

Synthesis of the Peptides

The peptides useful in the methods of the present invention may bechemically synthesized by any of the methods well known in the art.Suitable methods for synthesizing the protein include, for example thosedescribed by Stuart and Young in “Solid Phase Peptide Synthesis,” SecondEdition, Pierce Chemical Company (1984), and in “Solid Phase PeptideSynthesis,” Methods Enzymol. 289, Academic Press, Inc, New York (1997).

Modes of Administration

The peptide useful in the methods of the present invention isadministered to a mammal in an amount effective in reducing the numberof mitochondria undergoing, or preventing, MPT. The effective amount isdetermined during pre-clinical trials and clinical trials by methodsfamiliar to physicians and clinicians.

An effective amount of a peptide useful in the methods of the presentinvention, preferably in a pharmaceutical composition, may beadministered to a mammalian need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds.

The peptide may be administered systemically or locally. In oneembodiment, the peptide is administered intravenously. For example, thearomaticcationic peptides useful in the methods of the present inventionmay be administered via rapid intravenous bolus injection. Preferably,however, the peptide is administered as a constant rate intravenousinfusion.

The peptide can be injected directly into coronary artery during, forexample, angioplasty or coronary bypass surgery, or applied ontocoronary stents.

The peptide may also be administered orally, topically, intranasally,intramuscularly, subcutaneously, or transdermally. In a preferredembodiment, transdermal administration of the aromatic-cationic peptidesby methods of the present invention is by iontophoresis, in which thecharged peptide is delivered across the skin by an electric current.

Other routes of administration include intracerebroventricularly orintrathecally. Intracerebroventiculatly refers to administration intothe ventricular system of the brain. Intrathecally refers toadministration into the space under the arachnoid membrane of the spinalcord. Thus intracerebroventricular or intrathecal administration may bepreferred for those diseases and conditions which affect the organs ortissues of the central nervous system. In a preferred embodiment,intrathecal administration is used for traumatic spinal cord injury.

The peptides useful in the methods of the invention may also beadministered to mammals by sustained release, as is known in the art.Sustained release administration is a method of drug delivery to achievea certain level of the drug over a particular period of time. The leveltypically is measured by serum or plasma concentration.

Any formulation known in the art of pharmacy is suitable foradministration of the aromatic-cationic peptides useful in the methodsof the present invention. For oral administration, liquid or solidformulations may be used. Some examples of formulations include tablets,gelatin capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like. The peptides can be mixed with a suitablepharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art. Examples of carriers and excipients includestarch, milk, sugar, certain types of clay, gelatin, lactic acid,stearic acid or salts thereof, including magnesium or calcium stearate,talc, vegetable fats or oils, gums and glycols.

For systemic, intracerebroventricular, intrathecal, topical, intranasal,subcutaneous, or transdermal administration, formulations of thearomatic-cationic peptides useful in the methods of the presentinventions may utilize conventional diluents, carriers, or excipientsetc., such as are known in the art can be employed to deliver thepeptides. For example, the formulations may comprise one or more of thefollowing: a stabilizer, a surfactant, preferably a nonionic surfactant,and optionally a salt and/or a buffering agent. The peptide may bedelivered in the form of an aqueous solution, or in a lyophilized form.

The stabilizer may, for example, be an amino acid, such as for instance,glycine; or an oligosaccharide, such as for example, sucrose, tetralose,lactose or a dextran. Alternatively, the stabilizer may be a sugaralcohol, such as for instance, mannitol; or a combination thereof.Preferably the stabilizer or combination of stabilizers constitutes fromabout 0.1% to about 10% weight for weight of the peptide.

The surfactant is preferably a nonionic surfactant, such as apolysorbate. Some examples of suitable surfactants include Tween20,Tween80; a polyethylene glycolor a polyoxyethylene polyoxypropyleneglycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10%(w/v).

The salt or buffering agent may be any salt or buffering agent, such asfor example, sodium chloride, or sodium/potassium phosphate,respectively. Preferably, the buffering agent maintains the pH of thepharmaceutical composition in the range of about 5.5 to about 7.5. Thesalt and/or buffering agent is also useful to maintain the osmolality ata level suitable for administration to a human or an animal. Preferablythe salt or buffering agent is present at a roughly isotonicconcentration of about 150 mM to about 300 mM.

The formulations of the peptides useful in the methods of the presentinvention may additionally contain one or more conventional additive.Some examples of such additives include a solubilizer such as, forexample, glycerol; an antioxidant such as for example, benzalkoniumchloride (a mixture of quaternary ammonium compounds, known as “quats”),benzyl alcohol, chloretone or chlorobutanol; anaesthetic agent such asfor example a morphine derivative; or an isotonic agent etc., such asdescribed above. As a further precaution against oxidation or otherspoilage, the pharmaceutical compositions may be stored under nitrogengas in vials sealed with impermeable stoppers.

The mammal can be any mammal, including, for example, farm animals, suchas sheep, pigs, cows, and horses; pet animals, such as dogs and cats;laboratory animals, such as rats, mice and rabbits In a preferredembodiment, the mammal is a human.

EXAMPLES Example 1 [Dmt¹]DALDA Penetrates Cell Membrane

The cellular uptake of [³H] [Dmt¹]DALDA was studied using a humanintestinal epithelial cell line (Caco-2), and confirmed with SH-SY5Y(human neuroblastoma cell), HEK293 (human embryonic kidney cell) andCRFK cells (kidney epithelial cell). Monolayers of cells were grown on12-well plates (5×10⁵ cells/well) coated with collagen for 3 days. Onday 4, cells were washed twice with pre-wanned HBSS, and then incubatedwith 0.2 ml of HBSS containing either 250 nM [³H][Dmt¹]DALDA at 37° C.or 4° C. for various times up to 1 h.

[³H][Dmt¹]DALDA was observed in cell lysate as early as 5 min, andsteady state levels were achieved by 30 min. The total amount of[³H][Dmt¹]DALDA recovered in the cell lysate after 1 h incubationrepresented about 1% of the total drug. The uptake of [³H][Dmt¹]DALDAwas slower at 4° C. compared to 37° C., but reached 76.5% by 45 min and86.3% by 1 h. The internalization of [³H][Dmt¹]DALDA was not limited toCaco-2 cells, but was also observed in SH-SY5Y, HEK293 and CRFK cells.The intracellular concentration of [Dmt¹]DALDA was estimated to beapproximately 50 times higher than extracellular concentration.

In a separate experiment, cells were incubated with a range of[Dmt¹]DALDA concentrations (1 μM-3 mM) for 1 h at 37° C. At the end ofthe incubation period, cells were washed 4 times with HBSS, and 0.2 mlof 0.1N NaOH with 1% SDS was added to each well. The cell contents werethen transferred to scintillation vials and radioactivity counted. Todistinguish between internalized radioactivity from surface-associatedradioactivity, an acid-wash step was included. Prior to cell lysis,cells were incubated with 0.2 ml of 0.2 M acetic acid/0.05 M NaCl for 5min on ice.

The uptake of [Dmt¹]DALDA into Caco-2 cells was confirmed by confocallaser scanning microscopy (CLSM) using a fluorescent analog of[Dmt¹]DALDA (Dmt-D-Arg-Phe-dnsDap-NH₂; wherednsDap=β-dansyl-1-α,β-diaminopropionic acid). Cells were grown asdescribed above and were plated on (35 mm) glass bottom dishes (MatTekCorp., Ashland, Mass.) for 2 days. The medium was then removed and cellswere incubated with 1 ml of HBSS containing 0.1 μM to 1.0 μM of thefluorescent peptide analog at 37° C. for 1 h. Cells were then washedthree times with ice-cold HBSS and covered with 200 μl of PBS, andmicroscopy was performed within 10 min at room temperature using a Nikonconfocal laser scanning microscope with a C-Apochromat 63×/1.2 W corrobjective. Excitation was performed at 340 nm by means of a UV laser,and emission was measured at 520 nm. For optical sectioning inz-direction, 5-10 frames with 2.0 μm were made.

CLSM confirmed the uptake of fluorescent Dmt-D-Arg-Phe-dnsDap-NH₂ intoCaco-2 cells after incubation with 0.1 μM [Dmt¹,DnsDap⁴]DALDA for 1 h at37° C. The uptake of the fluorescent peptide was similar at 37° C. and4° C. The fluorescence appeared diffuse throughout the cytoplasm but wascompletely excluded from the nucleus.

Example 2 Targeting of [Dmt¹]DALDA to Mitochondria

To examine the subcellular distribution of [Dmt¹]DALDA, the fluorescentanalog, [Dmt¹, AtnDap⁴]DALDA (Dmt-D-Arg-Phe-atnDap-NH₂; whereatn=β-anthraniloyl-1-α,β,-diamino-propionic acid), was prepared. Theanalog contained β-anthraniloyl-1-α,β-diaminopropionic acid in place ofthe lysine reside at position 4. The cells were grown as described inExample 1 and were plated on (35 mm) glass bottom dishes (MatTek Corp.,Ashland, Mass.) for 2 days. The medium was then removed and cells wereincubated with 1 ml of HBSS containing 0.1 μM of [Dmt¹, AtnDap⁴]DALDA at37° C. for 15 min to 1 h.

Cells were also incubated with tetramethylrhodamine methyl ester (TMRM,25 nM), a dye for staining mitochondria, for 15 min at 37° C. Cells werethen washed three times with ice-cold HBSS and covered with 200 μl ofPBS, and microscopy was performed within 10 min at room temperatureusing a Nikon confocal laser scanning microscope with a C-Apochromat63×/1.2 W corr objective.

For [Dmt¹, AtnDap⁴]DALDA, excitation was performed at 350 nm by means ofa UV laser, and emission was measured at 520 nm. For TMRM, excitationwas performed at 536 nm, and emission was measured at 560 nm.

CLSM showed the uptake of fluorescent [Dmt¹, AtnDap⁴]DALDA into Caco-2cells after incubation for as little as 15 min at 37° C. The uptake ofdye was completely excluded from the nucleus, but the blue dye showed astreaky distribution within the cytoplasm. Mitochondria were labeled redwith TMRM. The distribution of [Dmt¹, AtnDap⁴]DALDA to mitochondria wasdemonstrated by the overlap of the [Dmt¹, AtnDap⁴]DALDA distribution andthe TMRM distribution.

Example 3 Uptake of [Dmt¹]DALDA into Mitochondria

To isolate mitochondria from mouse liver, mice were sacrificed bydecapitation. The liver was removed and rapidly placed into chilledliver homogenization medium. The liver was finely minced using scissorsand then homogenized by hand using a glass homogenizer.

The homogenate was centrifuged for 10 min at 1000×g at 4° C. Thesupernatant was aspirated and transferred to polycarbonate tubes andcentrifuged again for 10 min. at 3000×g, 4° C. The resulting supernatantwas removed, and the fatty lipids on the side-wall of the tube werecarefully wiped off.

The pellet was resuspended in liver homogenate medium and thehomogenization repeated twice. The final purified mitochondrial pelletwas resuspended in medium. Protein concentration in the mitochondrialpreparation was determined by the Bradford procedure.

Approximately 1.5 mg mitochondria in 400 μl buffer was incubated with[³H][Dmt¹]DALDA for 5-30 min at 37° C. The mitochondria were thencentrifuged down and the amount of radioactivity determined in themitochondrial fraction and buffer fraction. Assuming a mitochondrialmatrix volume of 0.7 μl/mg protein (Lim et al., J Physiol, 545:961-974,2002), the concentration of [³H][Dmt¹]DALDA in mitochondria was found tobe 200 times higher than in the buffer. Thus [Dmt¹]DALDA is concentratedin mitochondria.

Based on these data, the concentration of [Dmt¹]DALDA in mitochondriawhen the isolated guinea pig hearts were perfused with [Dmt¹]DALDA canbe estimated:

Concentration of [Dmt¹]DALDA in coronary perfusate 0.1 μM Concentrationof [Dmt¹]DALDA in myocyte  5 μM Concentration of [Dmt¹]DALDA inmitochondria 1.0 mM

Example 4 Accumulation of [Dmt¹]DALDA by Isolated Mitochondria (FIG. 1)

To further demonstrate that [Dmt¹]DALDA is selectively distributed tomitochondria, we examined the uptake of [Dmt¹, AtnDap⁴]DALDA and[³H][Dmt¹]DALDA into isolated mouse liver mitochondria. The rapid uptakeof [Dmt¹, AtnDap⁴]DALDA was observed as immediate quenching of itsfluorescence upon addition of mitochondria (FIG. 1 A). Pretreatment ofmitochondria with FCCP (carbonyl cyanidep-(trifluoromethoxy)-phenylhydrazone), an uncoupler that results inimmediate depolarization of mitochondria, only reduced [Dmt¹,AtnDap⁴]DALDA uptake by <20%. Thus uptake of [Dmt¹, AtnDap⁴]DALDA wasnot potential-dependent.

To confirm that the mitochondrial targeting was not an artifact of thefluorophore, we also examined mitochondrial uptake of [³H][Dmt¹]DALDA.Isolated mitochondria were incubated with [³H] [Dmt¹]DALDA andradioactivity determined in the mitochondrial pellet and supernatant.The amount of radioactivity in the pellet did not change from 2 min to 8min. Treatment of mitochondria with FCCP only decreased the amount of[³H][Dmt¹]DALDA associated with the mitochondrial pellet by ˜20% (FIG.1B).

The minimal effect of FCCP on [Dmt¹]DALDA uptake suggested that[Dmt¹]DALDA was likely to be associated with mitochondrial membranes orin the intermembrane space rather than in the matrix. We next examinedthe effect of mitochondrial swelling on the accumulation of [Dmt¹,AtnDap⁴]DALDA in mitochondria by using alamethicin to induce swellingand rupture of the outer membrane. Unlike TMRM, the uptake of [Dmt¹,AtnDap⁴]DALDA was only partially reversed by mitochondrial swelling(FIG. 1C). Thus, [Dmt¹]DALDA is associated with mitochondrial membranes.

Example 5 [Dmt¹]DALDA does not Alter Mitochondrial Respiration orPotential (FIG. 1D)

The accumulation of [Dmt¹]DALDA in mitochondria did not altermitochondrial function. Incubating isolated mouse liver mitochondriawith 100 μM [Dmt¹]DALDA did not alter oxygen consumption during state 3or state 4, or the respiratory ratio (state 3/state 4) (6.2 versus 6.0).Mitochondrial membrane potential was measured using TMRM (FIG. 1D)Addition of mitochondria resulted in immediate quenching of the TMRMsignal which was readily reversed by the addition of FCCP, indicatingmitochondrial depolarization. The addition of Ca²⁺ (150 μM) resulted inimmediate depolarization followed by progressive loss of quenchingindicative of MPT. Addition of [Dmt¹]DALDA alone, even at 200 μM, didnot cause mitochondrial depolarization or MPT.

Example 6 [Dmt¹]DALDA Protects Against MPT Induced by Ca²⁺ and3-Nitropropionic Acid (FIG. 2)

In addition to having no direct effect on mitochondrial potential,[Dmt¹]DALDA was able to protect against MPT induced by Ca²⁺ overload.Pretreatment of isolated mitochondria with [Dmt¹]DALDA (10 μM) for 2 minprior to addition of Ca²⁺ resulted only in transient depolarization andprevented onset of MPT (FIG. 2A), [Dmt¹]DALDA dose-dependently increasedthe tolerance of mitochondria to cumulative Ca²⁺ challenges. FIG. 2Bshows that [Dmt¹]DALDA increased the number of Ca²⁺ additions thatisolated mitochondria could tolerate prior to MPT.

3-Nitropropionic acid (3NP) is an irreversible inhibitor of succinatedehydrogenase in complex II of the electron transport chain. Addition of3NP (1 mM) to isolated mitochondria caused dissipation of mitochondrialpotential and onset of MPT (FIG. 2C). Pretreatment of mitochondria with[Dmt¹]DALDA dose-dependently delayed the onset of MPT induced by 3NP(FIG. 2C).

To demonstrate that [Dmt¹]DALDA can penetrate cell membranes and protectagainst mitochondrial depolarization elicited by 3NP, Caco-2 cells weretreated with 3NP (10 mM) in the absence or presence of [Dmt¹]DALDA (0.1μM) for 4 h, and then incubated with TMRM and examined under LSCM. Incontrol cells, the mitochondria are clearly visualized as fine streaksthroughout the cytoplasm. In cells treated with 3NP, the TMRMfluorescence was much reduced, suggesting generalized depolarization. Incontrast, concurrent treatment with [Dmt¹]DALDA protected againstmitochondrial depolarization caused by 3NP.

Example 7 [Dmt¹]DALDA Protects Against Mitochondrial Swelling andCytochrome c release

MPT pore opening results in mitochondrial swelling. We examined theeffects of [Dmt¹]DALDA on mitochondrial swelling by measuring reductionin absorbance at 540 nm (A₅₄₀). The mitochondrial suspension was thencentrifuged and cytochrome c in the mitochondrial pellet and supernatantdetermined by a commercially-available ELISA kit. Pretreatment ofisolated mitochondria with SS-02 inhibited swelling (FIG. 3A) andcytochrome c release (FIG. 3B) induced by Ca²⁺ overload. Besidespreventing MPT induced by Ca²⁺ overload, SS-02 also preventedmitochondrial swelling induced by MPP⁺ (1-methyl-4-phenylpyridium ion),an inhibitor of complex I of the mitochondrial electron transport chain(FIG. 3C).

Example 8 D-Arg-Dmt-Lys-Phe-NH₂ (SS-31) can Protect Against MPT,Mitochondrial Swelling and Cytochrome c Release

The non-opioid peptide SS-31 has the same ability to protect against MPT(FIG. 4A), mitochondrial swelling (FIG. 4B), and cytochrome c release(FIG. 4C), induced by Ca²⁺. The methods for study are as described abovefor SS-02. In this example, mitochondrial swelling was measured usinglight scattering monitored at 570 nm.

Example 9 [Dmt¹]DALDA (SS-02) and D-Arg-Dmt-Lys-Phc-NH₂ (SS-31) ProtectsAgainst Ischemia-Reperfusion-Induced Myocardial Stunning

Guinea pig hearts were rapidly isolated, and the aorta was cannulated insitu and perfused in a retrograde fashion with an oxygenatedKrebs-Henseleit solution (pH 7.4) at 34° C. The heart was then excised,mounted on a modified Langendorff perfusion apparatus, and perfused atconstant pressure (40 cm H₂O). Contractile force was measured with asmall hook inserted into the apex of the left ventricle and the silkligature tightly connected to a force-displacement transducer. Coronaryflow was measured by timed collection of pulmonary artery effluent.

Hearts were perfused with buffer, [Dmt¹]DALDA (SS-02) (100 nM) orD-Arg-Dmt-Lys-Phe-NH₂ (SS-31) (1 nM) for 30 min and then subjected to 30min of global ischemia. Reperfusion was carried out with the samesolution used prior to ischemia.

Two-way ANOVA revealed significant differences in contractile force(P<0.001), heart rate (P=0.003), and coronary flow (P<0.001) among thethree treatment groups. In the buffer group, contractile force wassignificantly lower during reperfusion compared with before ischemia(FIG. 5). Both SS-02 and SS-31 treated hearts tolerated ischemia muchbetter than buffer-treated hearts (FIG. 5). In particular, SS-31provided complete inhibition of cardiac stunning. In addition, coronaryflow is well-sustained throughout reperfusion and there was no decreasein heart rate.

Example 10 [Dmt¹]DALDA (SS-02) Enhances Organ Preservation

For heart transplantation, the donor heart is preserved in acardioplegic solution during transport. The preservation solutioncontains high potassium which effectively stops the heart from beatingand conserve energy. However, the survival time of the isolated heart isstill quite limited.

We examined whether [Dmt¹]DALDA prolongs survival of organs. In thisstudy, [Dmt¹]DALDA was added to a commonly used cardioplegic solution(St. Thomas) to determine whether [[Dmt¹]DALDA enhances survival of theheart after prolonged ischemia (model of ex vivo organ survival).

Isolated guinea pig hearts were perfused in a retrograde fashion with anoxygenated Krebs-Henseleit solution at 34° C. After 30 min. ofstabilization, the hearts were perfused with a cardioplegic solution CPS(St. Tohomas) with or without [Dmt¹]DALDA at 100 nM for 3 min. Globalischemia was then induced by complete interruption of coronary perfusionfor 90 min. Reperfusion was subsequently carried out for 60 min. withoxygenated Krebs-Henseleit solution. Contractile force, heart rate andcoronary flow were monitored continuously throughout the experiment.

The addition of [Dmt¹]DALDA to cardioplegic solution significantlyenhanced contractile function (FIG. 6) after prolonged ischemia.

Example 11 D-Arg-Dmt-Lys-Phe-NH₂ (SS-31) Prevented tBHP-InducedMitochondrial Depolarization

To investigate whether SS-31 prevents mitochondria) depolarizationcaused by tBHP, N2A cells were treated with 50 μM tBHP for 6 h.Treatment with tBHP resulted in a dramatic loss of mitochondrialpotential. Fluorescence intensity of TMRM (red), a cationic indicatorthat is taken up into mitochondria in a potential dependent manner, wassignificantly lower in cells treated with 50 μM tBHP (FIG. 7B), and thiswas completely blocked by concurrent treatment with 1 nM SS-31 (FIG.7C).

Example 12 D-Arg-Dmt-Lys-Phe-NH₂ (SS-31) and Phe-D-Arg-Phe-Lys-NH₂(SS-20) Prevents Myocardial Infarction in Rats MethodsInfarction/Reperfusion

Adult male Sprague-Dawley rats (F344 strain, National Institute ofAging, maintained by Harian Sprague-Dawley Inc.), weighting between 285and 425 gm, were used. Twenty-four rats (n=8/group) were randomlyassigned to one of three groups: (1) Control group was given 0.4 ml ofsaline as an intra-peritoneal (IP) injection 30 minutes before theligation of left anterior descending coronary artery, followed by sameIP dose injection 5 minutes before reperfusion; (2) SS-31 group wastreated with SS-31 (3 mg/kg dissolved in 0.4 ml of saline) as an IPinjection 30 minutes before the ligation followed by same IP doseinjection as maintenance 5 minutes before reperfusion; and (3) SS-20group was treated with SS-20 (3 mg/kg dissolved in 0.4 ml of saline) asan IP injection 30 minutes before the ligation followed by same dose IPinjection as maintenance just 5 minutes before reperfusion. Also,Sham-operated rats (n=3) were used to account for possible effectsrelated to the surgical protocol. All procedures were performed in ablinded manner, with the groups assigned letters and their identitiesunknown to the operators. Likewise, the two independent investigatorsanalyzing the data were blind to the treatment assignments.

After anesthesia with ketamine (90 mg/kg IP) and xylazine (4 mg/kg IP),a tracheotomy was performed, and the rat was intubated with polyethylenetube and ventilated (Harvard Rodent Ventilator model 683) with room airand a tidal volume of 0.65 ml/100 gm of body weight at 90 breaths perminute. Body temperature was maintained at 37° C. by using a heatedoperating table. The internal jugular vein was surgically exposed and apolyethylene tube was inserted for Evans blue dye solution injection.Peripheral limb electrodes were inserted subcutaneously andelectrocardiogram was monitored throughout the procedure.

The chest was opened by left thoracotomy at the fourth intercostal spaceto expose the heart. The pericardium was removed, and the left atrialappendage was gently moved to reveal the location of the left coronaryartery. The vein descending along the septum of the heart was used asthe marker for the left coronary artery. A suture ligature (7.0 Prolene)along with a snare occluder was placed around the vein and left coronaryartery close to the place of origin. The left anterior descendingcoronary artery was occluded by applying tension to the sling through apolyethylene 10 tubing and clamping. Successful occlusion was confirmedby elevation of the ST segment or the presence of deep S wave on the ECGand by cyanosis of the anterior wall of the left ventricle. Sixtyminutes after occlusion, the snare occluder was released and reperfusionof the myocardium was visually confirmed. The heart was then reperfusedfor sixty minutes. The heart was arrested in diastole with an overdoseof KCl and rapidly excised at the end of the experiment. Thesham-operated control group was subjected to thoracotomy and passage ofa silk ligature around the left coronary artery without ligation.

Determination of Area at Risk and Area of Infarction

At the end of reperfusion, the left coronary artery was brieflyre-occluded and Evans blue dye solution (2 ml of 2%, Sigma) was slowlyinjected into the jugular vein to distinguish the perfused area (bluestaining) from the area at risk (no staining). The excised hearts werecut parallel to the atrioventricular groove into 5 slices (˜1 mm thick)from base to apex. After removing all atrial and right ventricletissues, all slices were scanned. The slices were incubated in a 2%solution of triphenyl-tetrazolium chloride (TTC, Sigma) in phosphatebuffer for 20 min at 37° C. and pH of 7.4, and then immersed in 10%buffered formaldehyde for 14 days to distinguish the infarct area(unstained) from the viable myocardium (brick red staining). A prolongedformalin fixation was used to make infarct border zones easier tovisualize. After a 14-day formalin immersion, the slices were scannedagain and all scanned areas were quantified with NIH Imagesoftware. Areaat risk (AAR) was expressed as a percentage of the left ventricle (LV).Area of infarction (AI) was calculated as a percentage of the AAR.

Assessment of Arrhythmias

An ECG was recorded continuously from a standard lead II (AC AMP 700)inserted into the limbs of all rats. The ECG was printed at 25mm/second. The cardiac arrhythmias were monitored and assessed inaccordance with the Lambeth Convention. The assessment was performed ina blinded manner using the original paper recordings. A validated scorewas used to quantify the severity of cardiac arrhythmias. The scoreconsisted of six types: 0, no ventricular extrasystoles (VES),ventricular tachycardia (VT) or ventricular fibrillation (VF); 1=VES;2=one to five episodes of VT (more than four coupled VES); 3=more thanfive episodes of VT and/or one VF; 4=two to five episodes of VF; 5=morethan five episodes of VF.

Statistical Analysis

All values are expressed as mean±standard error of mean (S.E.M.).Statistical analyses were performed using SPSS version 10. Differencesamong groups in body weight, heart rate, arrhythinia, area at risk as apercentage of the left ventricle, and area of infarction as a percentageof the area at risk were analyzed using ANOVA. If significantdifferences were detected, comparisons between the control group and the2 treatment groups were conducted using the Mann-Whitney U-test. Atwo-tailed p<0.05 was regarded as significant.

Results Risk Areas and Infarct Sizes

Representative slices of left ventricle form control, SS-31 and SS-20groups are shown in FIG. 8. The AAR/LV ratio was similar among the threegroups (52.1±2.5% in the control group, 55.9±1.4%, p=0.38 in the SS-31group, and 52±2.1%, p=0.2 in the SS-20 group; FIG. 9). However, theAI/AAR ratio (FIG. 10) was significantly smaller in the SS-31 group(53.9±1.1%, p<0.01), in the SS-20 group (47.1±1.4%, p<0.01) than in thecontrol group (59.9±1). Meanwhile, AAR/LV ratio of sham group was50.3±4.6% (p=0.78 vs control) and Al/AAR ratio of sham group was3.7±3.7% (p<0.05 vs control).

Heart Rate and Cardiac Arrhythmias

Compared with controls, there were no significant differences in bodyweight and heart rate in any of the two groups during the study period(Table 3). Almost all arrhythmias occurred between 2 and 15 min aftercoronary occlusion. These rats with arrhythmias showed isolatedventricular extrasystole or nonsustained ventricular tachycardia (VT),but not ventricular fibrillation. They were transient and recoveredwithout therapy. Severity and occurrence rate of cardiac arrhythmias inSS-31 group (5 points, p<0.05) and SS-20 group (3 points, p<0.005)showed a significant reduction compared with control (13 points) duringthe entire study period.

TABLE 3 Body weight of all rats and heart rate during the course of theexperiment. Heart Rate Body Open weight Baseline chest Ischemia IschemiaReperfusion Reperfusion Control 351 ± 18 246 ± 4 212 ± 9 186 ± 10 166 ±8  176 ± 16 161 ± 4  (n = 8) SS-31 340 ± 12  257 ± 10 236 ± 8 184 ± 14169 ± 15 161 ± 12 164 ± 13 (n = 8) SS-20 349 ± 18 255 ± 5  211 ± 10 191± 17 184 ± 15 182 ± 16 183 ± 12 (n + 8) All values are expressed asmeans ± S.E.M. There was no difference among study groups (P < 0.05).

1. A method of reducing the number of mitochondria undergoingmitochondrial permeability transition (MPT), or preventing mitochondrialpermeability transitioning in a mammal in need thereof, the methodcomprising administering to the mammal an effective amount of anaromatic-cationic peptide having: (a) at least one net positive charge;(b) a minimum of four amino acids; (c) a maximum of about twenty aminoacids; (d) a relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) wherein3p_(m) is the largest number that is less than or equal to r+1; and (e)a relationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1, except that when a is 1,p_(t) may also be
 1. 2. The method according to claim 1, wherein 2 p_(m)is the largest number that is less than or equal to r+1.
 3. The methodaccording to claim 1, wherein a is equal to p_(t).
 4. The methodaccording to claim 1, wherein the peptide has a minimum of two positivecharges.
 5. The method according to claim 1, wherein the peptide has aminimum of three positive charges.
 6. The method according to claim 1,wherein the peptide is water soluble.
 7. The method according to claim1, wherein the peptide comprises one or more D-amino acids.
 8. Themethod according to claim 1, wherein the C-terminal carboxyl group ofthe amino acid at the C-terminus is amidated.
 9. The method according toclaim 1, wherein the peptide comprises one or more non-naturallyoccurring amino acids.
 10. The method according to claim 1, wherein thepeptide has a minimum of four amino acids.
 11. The method according toclaim 1, wherein the peptide has a maximum of about twelve amino acids.12. The method according to claim 1, wherein the peptide has a maximumof about nine amino acids.
 13. The method according to claim 1, whereinthe peptide has a maximum of about six amino acids.
 14. The methodaccording to claim 1, wherein the peptide has mu-opioid receptor agonistactivity.
 15. The method according to claim 1, wherein the peptide doesnot have mu-opioid receptor agonist activity.
 16. The method accordingto claim 1, wherein the mammal is a human.